Characterization of the proteome and metabolome of human liver sinusoidal endothelial-like cells derived from induced pluripotent stem cells.

The liver is a complex organ composed of several cell types organized hierarchically. Among these, liver sinusoidal endothelial cells (LSECs) are specialized vascular cells known to interact with hepatocytes and hepatic stellate cells (HSCs), and to be involved in the regulation of important hepatic processes in healthy and pathological situations. Protocols for the differentiation of LSECs from human induced pluripotent stem cells, hiPSCs, have been proposed and in-depth analysis by transcriptomic profiling of those cells has been performed. In the present work, an extended analysis of those cells in terms of proteome and metabolome has been implemented. The proteomic analysis confirmed the expression of important endothelial markers and pathways. Among them, the expression of patterns typical of LSECs such as PECAM1, VWF, LYVE1, STAB1 (endothelial markers), CDH13, CDH5, CLDN5, ICAM1, MCAM-CD146, ICAM2, ESAM (endothelial cytoskeleton), NOSTRIN, NOS3 (Nitric Oxide endothelial ROS), ESM1, ENG, MMRN2, THBS1, ANGPT2 (angiogenesis), CD93, MRC1 (mannose receptor), CLEC14A (C-type lectin), CD40 (antigen), and ERG (transcription factor) was highlighted. Besides, the pathway analysis revealed the enrichment of the endocytosis, Toll-like receptor, Nod-like receptor, Wnt, Apelin, VEGF, cGMP-PCK, and PPAR related signaling pathways. Other important pathways such as vasopressin regulated water reabsorption, fluid shear stress, relaxin signaling, and renin secretion were also highlighted. At confluence, the metabolome profile appeared consistent with quiescent endothelial cell patterns. The integration of both proteome and metabolome datasets revealed a switch from fatty acid synthesis in undifferentiated hiPSCs to a fatty oxidation in LSECs and activation of the pentose phosphate pathway and polyamine metabolism in hiPSCs-derived LSECs. In conclusion, the comparison between the signature of LSECs differentiated following the protocol described in this work, and data found in the literature confirmed the particular relevance of these cells for future in vitro applications.

miR-126-5p promotes retinal endothelial cell survival through SetD5 regulation in neurons.

MicroRNAs are key regulators of angiogenesis, as illustrated by the vascular defects observed in miR-126-deficient animals. The miR-126 duplex gives rise to two mature microRNAs (miR-126-3p and -5p). The vascular defects in these mutant animals were attributed to the loss of miR-126-3p but the role of miR-126-5p during normal angiogenesis in vivo remains unknown. Here, we show that miR-126-5p is expressed in endothelial cells but also by retinal ganglion cells (RGCs) of the mouse postnatal retina and participates in protecting endothelial cells from apoptosis during the establishment of the retinal vasculature. miR-126-5p negatively controls class 3 semaphorin protein (Sema3A) in RGCs through the repression of SetD5, an uncharacterized member of the methyltransferase family of proteins. In vitro, SetD5 controls Sema3A expression independently of its SET domain and co-immunoprecipitates with BRD2, a bromodomain protein that recruits transcription regulators onto the chromatin. Both SetD5 and BRD2 bind to the transcription start site and to upstream promoter regions of the Sema3a locus and BRD2 is necessary for the regulation of Sema3A expression by SetD5. Thus, neuronally expressed miR-126-5p regulates angiogenesis by protecting endothelial cells of the developing retinal vasculature from apoptosis.

Collagen Suprafibrillar Confinement Drives the Activity of Acidic Calcium-Binding Polymers on Apatite Mineralization.

Bone collagenous extracellular matrix provides a confined environment into which apatite crystals form. This biomineralization process is related to a cascade of events partly controlled by noncollagenous proteins. Although overlooked in bone models, concentration and physical environment influence their activities. Here, we show that collagen suprafibrillar confinement in bone comprising intra- and interfibrillar spaces drives the activity of biomimetic acidic calcium-binding polymers on apatite mineralization. The difference in mineralization between an entrapping dentin matrix protein-1 (DMP1) recombinant peptide (rpDMP1) and the synthetic polyaspartate validates the specificity of the 57-KD fragment of DMP1 in the regulation of mineralization, but strikingly without phosphorylation. We show that all the identified functions of rpDMP1 are dedicated to preclude pathological mineralization. Interestingly, transient apatite phases are only found using a high nonphysiological concentration of additives. The possibility to combine biomimetic concentration of both collagen and additives ensures specific chemical interactions and offers perspectives for understanding the role of bone components in mineralization.

Endothelial Cell Activation Is Regulated by Epidermal Growth Factor-like Domain 7 (Egfl7) during Inflammation.

Activation of the blood vessel endothelium is a critical step during inflammation. Endothelial cells stimulated by pro-inflammatory cytokines play an essential part in the adhesion and extravasation of circulating leukocytes into inflamed tissues. The endothelial egfl7 gene (VE-statin) represses endothelial cell activation in tumors, and prior observations suggested that it could also participate in the regulation of endothelial cell activation during inflammation. We show here that Egfl7 expression is strongly repressed in mouse lung endothelial cells during LPS- and TNFα-induced inflammation in vivo LPS have a limited effect on Egfl7 expression by endothelial cells in vitro, whereas the pro-inflammatory cytokine TNFα strongly represses Egfl7 expression in endothelial cells. TNFα regulates the egfl7 gene promoter through regions located between -7585 and -5550 bp ahead of the main transcription start site and via an NF-κB-dependent mechanism. Conversely, Egfl7 regulates the response of endothelial cells to TNFα by restraining the induced expression of intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), and E-selectin, resulting in a decreased adhesion of leukocytes onto endothelial cells stimulated by TNFα. Egfl7 regulates the expression of these adhesion molecules through the NF-κB and MEK/Erk pathways, in particular by preventing the proteasome-mediated degradation of IkBα both in non-activated endothelial cells and during activation. Egfl7 is thus an endogenous and constitutive repressor of blood vessel endothelial cell activation in normal and inflammatory conditions and participates in a loop of regulation of activation of these cells by pro-inflammatory cytokines.

Coculture model of a liver sinusoidal endothelial cell barrier and HepG2/C3a spheroids-on-chip in an advanced fluidic platform.

The liver is one of the main organs involved in the metabolism of xenobiotics and a key organ in toxicity studies. Prior to accessing the hepatocytes, xenobiotics pass through the hepatic sinusoid formed by liver sinusoidal endothelial cells (LSECs). The LSECs barrier regulates the kinetics and concentrations of the xenobiotics before their metabolic processing by the hepatocytes. To mimic this physiological situation, we developed an in vitro model reproducing an LSECs barrier in coculture with a hepatocyte biochip, using a fluidic platform. This technology made dynamic coculture and tissue crosstalk possible. SK-HEP-1 and HepG2/C3a cells were used as LSECs and as hepatocyte models, respectively. We confirmed the LSECs phenotype by measuring PECAM-1 and stabilin-2 expression levels and the barrier's permeability/transport properties with various molecules. The tightness of the SK-HEP-1 barrier was enhanced in the dynamic coculture. The morphology, albumin secretion, and gene expression levels of markers of HepG2/C3a were not modified by coculture with the LSECs barrier. Using acetaminophen, a well-known hepatotoxic drug, to study tissue crosstalk, there was a reduction in the expression levels of the LSECs markers stabilin-2 and PECAM-1, and a modification of those of CLEC4M and KDR. No HepG2/C3a toxicity was observed. The metabolisation of acetaminophen by HepG2/C3a monocultures and cocultures was confirmed. Although primary cells are required to propose a fully relevant model, the present approach highlights the potential of our system for investigating xenobiotic metabolism and toxicity.

Differential proteomic analysis of human glioblastoma and neural stem cells reveals HDGF as a novel angiogenic secreted factor.

Presence in glioblastomas of cancer cells with normal neural stem cell (NSC) properties, tumor initiating capacity, and resistance to current therapies suggests that glioblastoma stem-like cells (GSCs) play central roles in glioblastoma development. We cultured human GSCs endowed with all features of tumor stem cells, including tumor initiation after xenograft and radio-chemoresistance. We established proteomes from four GSC cultures and their corresponding whole tumor tissues (TTs) and from human NSCs. Two-dimensional difference gel electrophoresis and tandem mass spectrometry revealed a twofold increase of hepatoma-derived growth factor (HDGF) in GSCs as compared to TTs and NSCs. Western blot analysis confirmed HDGF overexpression in GSCs as well as its presence in GSC-conditioned medium, while, in contrast, no HDGF was detected in NSC secretome. At the functional level, GSC-conditioned medium induced migration of human cerebral endothelial cells that can be blocked by anti-HDGF antibodies. In vivo, GSC-conditioned medium induced neoangiogenesis, whereas HDGF-targeting siRNAs abrogated this effect. Altogether, our results identify a novel candidate, by which GSCs can support neoangiogenesis, a high-grade glioma hallmark. Our strategy illustrates the usefulness of comparative proteomic analysis to decipher molecular pathways, which underlie GSC properties.

Genes Involved in miRNA Biogenesis Are Not Downregulated in SARS-CoV-2 Infection.

miRNAs, small non-coding RNAs that regulate gene expression, are involved in various pathological processes, including viral infections. Virus infections may interfere with the miRNA pathway through the inhibition of genes involved in miRNA biogenesis. A reduction in the number and the levels of miRNAs expressed in nasopharyngeal swabs of patients with severe COVID-19 was lately observed by us, pointing towards the potential of miRNAs as possible diagnostic or prognostic biomarkers for predicting outcomes among patients with severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection. The objective of the present study was to investigate whether SARS-CoV-2 infection influences the expression levels of messenger RNAs (mRNAs) of key genes involved in miRNA biogenesis. mRNA levels of AGO2, DICER1, DGCR8, DROSHA, and Exportin-5 (XPO5) were measured by quantitative reverse-transcription polymerase chain reaction (RT-qPCR) in nasopharyngeal swab specimens from patients with COVID-19 and controls, as well as in cells infected with SARS-CoV-2 in vitro. Our data showed that the mRNA expression levels of AGO2, DICER1, DGCR8, DROSHA, and XPO5 were not significantly different in patients with severe COVID-19 when compared to patients with non-severe COVID-19 and controls. Similarly, the mRNA expression of these genes was not affected by SARS-CoV-2 infection in NHBE and Calu-3 cells. However, in Vero E6 cells, AGO2, DICER1, DGCR8, and XPO5 mRNA levels were slightly upregulated 24 h after infection with SARS-CoV-2. In conclusion, we did not find evidence for downregulation of mRNA levels of miRNA biogenesis genes during SARS-CoV-2 infection, neither ex vivo nor in vitro.

Investigation of the metabolomic crosstalk between liver sinusoidal endothelial cells and hepatocytes exposed to paracetamol using organ-on-chip technology.

Organ-on-chip technology is a promising in vitro approach recapitulating human physiology for the study of responses to drug exposure. Organ-on-chip cell cultures have paved new grounds for testing and understanding metabolic dose-responses when evaluating pharmaceutical and environmental toxicity. Here, we present a metabolomic investigation of a coculture of liver sinusoidal endothelial cells (LSECs, SK-HEP-1) with hepatocytes (HepG2/C3a) using advanced organ-on-chip technology. To reproduce the physiology of the sinusoidal barrier, LSECs were separated from hepatocytes by a membrane (culture insert integrated organ-on-chip platform). The tissues were exposed to acetaminophen (APAP), an analgesic drug widely used as a xenobiotic model in liver and HepG2/C3a studies. The differences between the SK-HEP-1, HepG2/C3a monocultures and SK-HEP-1/HepG2/C3a cocultures, treated or not with APAP, were identified from metabolomic profiles using supervised multivariate analysis. The pathway enrichment coupled with metabolite analysis of the corresponding metabolic fingerprints contributed to extracting the specificity of each type of culture and condition. In addition, we analysed the responses to APAP treatment by mapping the signatures with significant modulation of the biological processes of the SK-HEP-1 APAP, HepG2/C3a APAP and SK-HEP-1/HepG2/C3a APAP conditions. Furthermore, our model shows how the presence of the LSECs barrier and APAP first pass can modify the metabolism of HepG2/C3a. Altogether, this study demonstrates the potential of a "metabolomic-on-chip" strategy for pharmaco-metabolomic applications predicting individual response to drugs.

VE-statin/egfl7 regulates vascular elastogenesis by interacting with lysyl oxidases.

We previously characterized VE-statin/egfl7, a protein that is exclusively secreted by endothelial cells and modulates smooth muscle cell migration. Here, we show that VE-statin/egfl7 is the first known natural negative regulator of vascular elastogenesis. Transgenic mice, expressing VE-statin/egfl7 under the control of keratin-14 promoter, showed an accumulation of VE-statin/egfl7 in arterial walls where its presence correlated with an impaired organization of elastic fibres. In vitro, fibroblasts cultured in the presence of VE-statin/egfl7 were unable to deposit elastic fibres due to a deficient conversion of soluble tropoelastin into insoluble mature elastin. VE-statin/egfl7 interacts with the catalytic domain of lysyl oxidase (LOX) enzymes and, in endothelial cells, endogenous VE-statin/egfl7 colocalizes with LoxL2 and inhibits elastic fibre deposition. In contrast, mature elastic fibres are abundantly deposited by endothelial cells that are prevented from producing endogenous VE-statin/egfl7. We propose a model where VE-statin/egfl7 produced by endothelial cells binds to the catalytic domains of enzymes of the LOX family in the vascular wall, thereby preventing the crosslink of tropoelastin molecules into mature elastin polymers and regulating vascular elastogenesis.

The impact of macrophages on endothelial cells is potentiated by cycling hypoxia: Enhanced tumor inflammation and metastasis.

Cycling hypoxia (cyH), neo-angiogenesis, and tumor-associated macrophages are key features of the tumor microenvironment. In this study, we demonstrate that cyH potentiates the induction by unpolarized and M1-like macrophages of endothelial inflammatory phenotype and adhesiveness for monocytes and cancer cells. This process triggers a positive feedback loop sustaining tumor inflammation. This work opens the door for innovative therapeutic strategies to treat tumor inflammation and metastasis. In cancers, the interaction between macrophages and endothelial cells (ECs) regulates tumor inflammation and metastasis. These cells are both affected by cycling hypoxia (cyH), also called intermittent hypoxia, a feature of the tumor microenvironment. cyH is also known to favor tumor inflammation and metastasis. Nonetheless, the potential impact of cyH on the dialog between macrophages and ECs is still unknown. In this work, the effects of unpolarized, M1-like, and M2-like macrophages exposed to normoxia, chronic hypoxia (chH), and cyH on endothelial adhesion molecule expression, pro-inflammatory gene expression, and EC adhesiveness for monocytes and cancer cells were investigated. cyH increased the ability of unpolarized and M1-like macrophages to induce EC inflammation and to increase the expression of the EC endothelial adhesion molecule ICAM1, respectively. Unpolarized, M1-like, and M2-like macrophages were all able to promote EC adhesive properties toward cancer cells. Furthermore, the ability of macrophages (mostly M1-like) to shift EC phenotype toward one allowing cancer cell and monocyte adhesion onto ECs was potentiated by cyH. These effects were specific to cyH because they were not observed with chH. Together, these results show that cyH amplifies the effects of macrophages on ECs, which may promote tumor inflammation and metastasis.

Multi-Layered Human Blood Vessels-on-Chip Design Using Double Viscous Finger Patterning.

Blood vessel-on-a-chip models aim at reproducing vascular functions. However, very few efficient methods have been designed to address the need for biological replicates in medium- to high-throughput screenings. Here, vessels-on-chip were designed in polydimethylsiloxane-glass chips using the viscous finger patterning technique which was adapted to create channels with various internal diameters inside a collagen solution and to simultaneously seed cells. This method was refined to create blood vessels composed of two concentric, distinct, and closely appositioned layers of human endothelial and perivascular cells arranged around a hollow lumen. These approaches allowed the formation of structurally correct blood vessels-on-chips which were constituted of either only endothelial cells or of both cell types in order to distinguish the vascular barrier reactivity to drugs in the presence or not of perivascular cells. The established vessels showed a tight vascular barrier, as assessed by immunostaining of the adherens junctions, and were reactive to the natural vasopermeant thrombin and to inflammatory cytokines. The presence of perivascular cells markedly increased the tightness of the vascular barrier and lowered its response to thrombin. The design allowed us to simultaneously challenge in real-time several tens of 3D-reconstituted, multicellular blood vessels in a standard multiwell plate format suitable for high-throughput drug screening.

EGF repeats of epidermal growth factor­like domain 7 promote endothelial cell activation and tumor escape from the immune system.

The tumor blood vessel endothelium forms a barrier that must be crossed by circulating immune cells in order for them to reach and kill cancer cells. Epidermal growth factor­like domain 7 (Egfl7) represses this immune infiltration by lowering the expression levels of leukocyte adhesion receptors on the surface of endothelial cells. However, the protein domains involved in these properties are not completely understood. Egfl7 is structurally composed of the predicted EMI­, EGF­ and C­terminal domains. The present study aimed to investigate the roles of these different domains in tumor development by designing retroviruses coding for deletion mutants and then infecting 4T1 breast cancer cell populations, which consequently overexpressed the variants. By performing in vitro soft­agar assays, it was found that Egfl7 and its deletion variants did not affect cell proliferation or anchorage­independent growth. When 4T1 cells expressing either the wild­type Egfl7 protein or Egfl7 domain variants were implanted in mice, Egfl7 expression markedly promoted tumor development and deletion of the EGF repeats decreased the tumor growth rate. By contrast, deleting any other domain displayed no significant effect on tumor development. The overexpression of Egfl7 also decreased T cell and natural killer cell infiltration in tumors, as determined by immunofluorescence staining of tumor sections, whereas deletion of the EGF repeats inhibited this effect. Reverse transcription­quantitative PCR analysis of the mechanisms involved revealed that deleting the EGF repeats partially restored the expression levels of vascular cell adhesion molecule 1 and E­selectin, which were suppressed by overexpression of Egfl7 in endothelial cells in vitro. This resulted in a higher number of lymphocytes bound to HUVEC expressing Egfl7­ΔEGF compared with HUVEC expressing wild­type Egfl7, as assessed by fluorescent­THP­1 adhesion assays onto endothelial cells. Overall, the present study demonstrated that the EGF repeats may participate in the protumoral and anti­inflammatory effects of Egfl7.

IL-2 is inactivated by the acidic pH environment of tumors enabling engineering of a pH-selective mutein.

Cytokines interact with their receptors in the extracellular space to control immune responses. How the physicochemical properties of the extracellular space influence cytokine signaling is incompletely elucidated. Here, we show that the activity of interleukin-2 (IL-2), a cytokine critical to T cell immunity, is profoundly affected by pH, limiting IL-2 signaling within the acidic environment of tumors. Generation of lactic acid by tumors limits STAT5 activation, effector differentiation, and antitumor immunity by CD8+ T cells and renders high-dose IL-2 therapy poorly effective. Directed evolution enabled selection of a pH-selective IL-2 mutein (Switch-2). Switch-2 binds the IL-2 receptor subunit IL-2Rα with higher affinity, triggers STAT5 activation, and drives CD8+ T cell effector function more potently at acidic pH than at neutral pH. Consequently, high-dose Switch-2 therapy induces potent immune activation and tumor rejection with reduced on-target toxicity in normal tissues. Last, we show that sensitivity to pH is a generalizable property of a diverse range of cytokines with broad relevance to immunity and immunotherapy in healthy and diseased tissues.

EGFL7 regulates sprouting angiogenesis and endothelial integrity in a human blood vessel model.

Elucidating the mechanisms underlying sprouting angiogenesis and permeability should enable the development of more effective therapies for various diseases, including retinopathy, cancer, and other vascular disorders. We focused on epidermal growth factor-like domain 7 (EGFL7) which plays an important role in NOTCH signaling and in the organization of angiogenic sprouts. We developed an EGFL7-knockdown in vitro microvessel model and investigated the effect of EGFL7 at a tissue level. We found EGFL7 knockdown suppressed VEGF-A-induced sprouting angiogenesis accompanied by an overproduction of endothelial filopodia and reduced collagen IV deposition at the basal side of endothelial cells. We also observed impaired barrier function which reflected an inflammatory condition. Furthermore, our results showed that proper formation of adherens junctions and phosphorylation of VE-cadherin was disturbed. In conclusion, by using a 3D microvessel model we identified novel roles for EGFL7 in endothelial function during sprouting angiogenesis.

MAGP-1 and fibronectin control EGFL7 functions by driving its deposition into distinct endothelial extracellular matrix locations.

The extracellular matrix (ECM) is essential to provide mechanical support to tissues but is also a bioactive edifice which controls cell behavior. Cell signaling generated by ECM components through integrin-mediated contacts, modulates cell biological activity. In addition, by sequestrating or releasing growth factors, the ECM is an active player of physiological and pathological processes such as vascular development. EGFL7 is mainly expressed during blood vessel development and is deposited in the ECM after secretion by endothelial cells. While EGFL7 is known to control various endothelial cell molecular mechanisms [i.e., the repression of endothelial-derived lysyl oxidase (LOX) enzyme, the regulation of the Notch pathway, and the expression of leukocyte adhesion molecules and of RHOA by endothelial cells], it is not established whether EGFL7 functions when bound to the ECM. Here, we show that microfibrillar-associated glycoprotein-1 (MAGP-1) and fibronectin drive the deposition of EGFL7 into both fibers and individual aggregates in endothelial ECM. Although EGFL7 does not need to be docked into the ECM to control endothelial adhesion molecule expression, the ECM accumulation of EGFL7 is required for its regulation of LOX activity and of HEY2 expression along the Notch pathway. The interaction of EGFL7 with MAGP-1 is necessary for LOX activity repression by EGFL7 while it does not participate in the control of the Notch pathway by this protein. Altogether, this study highlights the roles played by EGFL7 in controlling various endothelial molecular mechanisms upon its localization and shows how the ECM can modulate its functions.

A Vascular Endothelial Growth Factor-Dependent Sprouting Angiogenesis Assay Based on an In Vitro Human Blood Vessel Model for the Study of Anti-Angiogenic Drugs.

Angiogenesis is the formation of new capillaries from pre-existing blood vessels and participates in proper vasculature development. In pathological conditions such as cancer, abnormal angiogenesis takes place. Angiogenesis is primarily carried out by endothelial cells, the innermost layer of blood vessels. The vascular endothelial growth factor-A (VEGF-A) and its receptor-2 (VEGFR-2) trigger most of the mechanisms activating and regulating angiogenesis, and have been the targets for the development of drugs. However, most experimental assays assessing angiogenesis rely on animal models. We report an in vitro model using a microvessel-on-a-chip. It mimics an effective endothelial sprouting angiogenesis event triggered from an initial microvessel using a single angiogenic factor, VEGF-A. The angiogenic sprouting in this model is depends on the Notch signaling, as observed in vivo. This model enables the study of anti-angiogenic drugs which target a specific factor/receptor pathway, as demonstrated by the use of the clinically approved sorafenib and sunitinib for targeting the VEGF-A/VEGFR-2 pathway. Furthermore, this model allows testing simultaneously angiogenesis and permeability. It demonstrates that sorafenib impairs the endothelial barrier function, while sunitinib does not. Such in vitro human model provides a significant complimentary approach to animal models for the development of effective therapies.

Egfl7 Represses the Vasculogenic Potential of Human Endothelial Progenitor Cells.

Egfl7 (VE-statin) is a secreted protein mostly specific to the endothelial lineage during development and in the adult and which expression is enhanced during angiogenesis. Egfl7 involvement in human postnatal vasculogenesis remains unresolved yet. Our aim was to assess Egfl7 expression in several angiogenic cell types originating from human bone marrow, peripheral blood, or cord blood. We found that only endothelial colony forming cells (ECFC), which are currently considered as the genuine endothelial precursor cells, expressed large amounts of Egfl7. In order to assess its potential roles in ECFC, Egfl7 was repressed in ECFC by RNA interference and ECFC angiogenic capacities were tested in vitro and in vivo. Cell proliferation, differentiation, and migration were significantly improved when Egfl7 was repressed in ECFC in vitro, whereas miR-126-3p levels remained unchanged. In vivo, repression of Egfl7 in ECFC significantly improved post-ischemic revascularization in a model of mouse hind-limb ischemia. In conclusion, ECFC are the sole postnatal angiogenic cells which express large amounts of Egfl7 and whose angiogenic properties are repressed by this factor. Thus, Egfl7 inhibition may be considered as a therapeutic option to improve ECFC-mediated postnatal vasculogenesis and to optimize in vitro ECFC expansion in order to develop an optimized cell therapy approach.

Bone morphogenic proteins are overexpressed in malignant melanoma and promote cell invasion and migration.

Malignant melanoma cells are known to have altered expression of growth factors compared with normal human melanocytes. These changes probably favor tumor growth and progression and influence the tumor environment. The induction of transforming growth factor beta1 (TGF-beta1), TGF-beta2, and TGF-beta3 expression in malignant melanoma has been reported before, whereas the expression of related bone morphogenic protein (BMP) molecules has not been analyzed in melanomas until now. Here, we show that BMP4 and BMP7 are up-regulated in nine melanoma cell lines, whereas BMP2 is overexpressed in only two of the analyzed cell lines. Immunohistochemistry of primary and metastatic melanoma also shows increased BMP4 and BMP7 expression compared with nevi. Promoter studies reveal that expression is controlled at the transcriptional level. The transcription factor Ets-1 was identified as a positive regulator for BMP4 expression. In order to determine the functional relevance of BMP expression in malignant melanoma, chordin-expressing cell clones and antisense BMP4 cell clones were generated. The clones in which BMP4 activity and expression are reduced show no changes in proliferation or in attachment-independent growth when compared with controls. However, a strong reduction of migratory and invasive properties was observed in these cells, suggesting that BMP4 promotes melanoma cell invasion and migration and therefore has an important role in the progression of malignant melanoma.

Co-delivery of the NKT agonist α-galactosylceramide and tumor antigens to cross-priming dendritic cells breaks tolerance to self-antigens and promotes antitumor responses.

Vaccines designed to abrogate the tolerance of tumor self-antigens and amplify cytotoxic CD8+ T cells (CTLs) have promise for the treatment of cancer. Type I natural killer (NKT) cells have attracted considerable interest in the cancer therapy field. In the current study, we have exploited the unique ability of NKT cells to serve as T-helper cells to license dendritic cells (DCs) for cross priming with the aim to generate efficient CTL antitumor responses. To this end, we designed a nanoparticle-based vaccine to target cross-priming DCs via the Clec9a endocytic pathway. Our results showed for the first time that simultaneous co-delivery of the NKT agonist α-galactosylceramide and tumor self-antigens (Trp2 and gp100) to CD8α+ DCs promotes strong antitumor responses in prophylactic and therapeutic settings (advanced solid tumor model in the mouse). We attributed the vaccine's therapeutic effects to NKT cells (but not to T-helper lymphocytes) and CD8+ T cells. Efficacy was correlated with an elevated ratio between tumor antigen-specific CD8+ T cells and regulatory CD4+ T lymphocytes within the tumor. The nanoparticle-based vaccine actively targeted human CLEC9A-expressing BDCA3+ DCs - the equivalent of murine cross-priming CD8α+ DCs - and induced a strong expansion of effector memory tumor self-antigen (Melan -A)-specific CD8+ T cells from peripheral blood mononuclear cells sourced from healthy donors and melanoma patients. Together, our result shed light on novel therapeutic approaches for controlling tumor development.